Project Summary/Abstract Non-erythroid hemoglobin production was first reported in macrophages almost 20 years ago. Since that time, hemoglobin has been found broadly expressed in a number of tissues under physiological and pathophysiological conditions including the endometrium and ovary. Surprisingly, very little is known about non- erythroid hemoglobin production both in terms of its transcriptional regulation and its functions. Some of the proposed functions for non-erythroid hemoglobin include: 1) oxygen transport/sequestration; 2) regulation of nitric oxide production/signaling; 3) reducing nitrosative stress; 4) iron transport; 5) antimicrobial actions; and 6) sequestration of reactive oxygen species (ROS, antioxidant properties). It is interesting to note that a functional placenta does not exist in the human until several weeks into the first trimester. In mice, the placenta develops and becomes functional around day 10 of pregnancy, which is half way through gestation. Mechanisms governing ROS and oxygen bioavailability at the pregnancy interface, particularly pre-placentation, are ill- defined. In erythroid tissue, hemoglobin and associated genes are regulated by a transcriptional complex that consists of transcription factor 3 (TCF3), LIM domain binding protein 1 (LDB1), GATA transcription factor 1 (GATA1), and Krueppel-like factor 1 (KLF1). Through mutagenesis studies in mice, our group recently established that two components of this TCF3/LDB1/GATA1/KLF1 transcriptional complex are essential for normal fertility in the female. Indeed, conditional uterine ablation of Tcf3 or Ldb1 results in infertility or severe subfertility, respectively. Furthermore, hemoglobin-?, hemoglobin-?, the rate limiting enzyme in heme production, aminolevulinic acid synthase 2 (previously thought to be exclusively restricted to the erythroid lineage), and several hemoglobin biosynthesis-dependent ribosomal proteins were all shown to be down regulated in Tcf3 and Ldb1 mutant uteri. Based on these findings, it is proposed that uterine-derived hemoglobin serves to tightly regulate local oxygen tension and prevent accumulation of ROS that would otherwise compromise normal embryonic/fetal development. The central hypothesis to be tested is that uterine-derived hemoglobin plays a fundamental role in the establishment and maintenance of pregnancy in mammals. This hypothesis will be tested by evaluating the consequences of conditionally ablating hemoglobin genes from the uterus during gestation. From a basic science standpoint, our studies are significant because they will provide meaningful information related to the transcriptional regulation and function(s) of non-erythroid hemoglobin that will likely be of broad interest to many areas of biology. From a clinical perspective, these studies will shed new light on possible ways to enhance in vitro fertilization outcomes and could be used to help explain idiopathic pregnancy failure, congenital birth defects, or some of the developmental origins of disease.